Our long-term goal is to understand, in detail, the mechanism of amyloid formation from various non-homologous amyioidogenic proteins, to elucidate the relationship between protein misfolding and amyloid diseases, and to develop therapeutic models for inhibition of amyloid formation. In this proposal, we focus on the study of early events along the pathway of human transthyretin (TTR) amyloid formation using computational approaches at the atomic level. We propose three specific aims: Aim 1: A computational study of the energy landscapes, conformational transition pathways, and concerted motions. It is highly likely that the stability and flexibility of monomedc TTR play crucial roles in the early steps of amyloid formation, thereby, it is essential to characterize the initial conformational changes of monomeric I-FR. We begin with simulations of the wild-type TTR monomer and its mutants to characterize their dynamic properties, map the energy landscapes, and search for likely conformational transition pathways connecting the native state and the intermediate states of TTR. By comparing the wild-type with the amyloidogenic mutants, we will provide insights on the differential amyloidogenesis. Aim 2: To study the pH dependence of structure, pH is a crucial factor of the condition which facilitates conformational changes. How does the solution pH influence the tetramedc and monomeric structure of TTR? We propose to perform simulations at constant pH. We will employ a novel algorithm to generate ensembles of protonation states satisfying Boltzmann distribution. By analyzing the population of each protonation state and the generated trajectories, we will provide information on the structural changes as a function of pH. Aim 3: To identify the most favorable binding mode of small molecules bound to TTR and design Imodifications of the ligand that optimize its most favorable binding mode. We propose to study the role of protein-small molecule interaction in stabilizing the tetrameric native state of TTR, to identify the most favorable ligand binding mode among the multiple binding modes with a novel free energy calculation method, and to optimize the ligands to enhance binding affinity. The proposed calculations will provide insight into the small-molecule strategy for intervening in the pathology of TTR related amyioid diseases.